Apoptosis plays a variety of important roles in the immune system, and it is the goal of this application to elucidate the central mechanisms of this form of cell death. A critical decision point in the process occurs when the mitochondrial outer membrane permeabilizes to release proteins from the intermembrane space. Two of these, cytochrome c and Smac/DIABLO regulate the activation of caspase proteases which in turn orchestrate the execution of the cell. This "mitochondria-caspase axis" is the major target for regulation of apoptosis and serves as the focus of the current application. Three fundamental aspects of this critical decision point in cell death will be addressed. In the first aim, we will examine the relationships between cytochrome c release and mitochondrial function in lymphoid cells during apoptosis. This will be achieved through the use of cells expressing cytochrome c-GFP and the application of imaging techniques we have developed. Changes in mitochondrial function can occur as a consequence of aging, disease, oncogenesis, or lymphocyte activation, and the impact of such changes on the timing and release of cytochrome c-GFP will be evaluated. The importance of cytochrome c in triggering caspase activation in lymphocyte apoptosis will be evaluated through the use of mice defective in Apaf-1. In the second aim we will examine the mitochondrial-caspase axis in death receptor-and granzyme B-mediated cytotoxicity. We will test the hypothesis that caspase activation is blocked by TAP proteins, such as XIAP, and that reversal of this effect by Smac (released from the mitochondria) can be necessary for caspase activation in response to death receptor or granzyme B signals. The ability of Bcl-2 to block such apoptosis will therefore depend on levels of the TAPs. We will also analyze the regulation of ubiquitination and proteasomal degradation of caspases by the Smac-IAP interaction, as XIAP is a ubiquitin ligase. In our third aim, we will determine the effects of caspases on mitochondrial function following mitochondrial outer membrane permeabilization. We have found that the mitochondria are among the earliest targets for activated caspases in the cell, and as a result mitochondrial function is lost. We will identify the relevant caspase targets in the mitochondria and explore the roles of these in the changes in mitochondrial function following caspase activation. The research program outlined in this application therefore focuses on the two way interaction between mitochondria and caspases during apoptosis, with special emphasis on cell death in lymphocytes. Our studies will elucidate how the central mechanisms of this mitochondrial-caspase axis control life/death decisions in cells.